Article Figures & Data
Figures
- Figure 1.
CD8+ T cells proliferate in the tumor microenvironment. A, C57BL/6 mice were injected subcutaneously with 2 × 106 B16.SIY tumor cells on day 0 and sacrificed at the indicated time points. One day prior to sacrifice, mice were injected intraperitoneally with BrdU (0.8 mg). Spleens and tumors of mice were analyzed via flow cytometry for BrdU incorporation in CD8+ T cells. Day 7, n = 5; day 10, n = 10; day 13, n = 10; day 16, n = 10; day 21, n = 5. Data pooled from 3 independent experiments, two-way ANOVA. B, Analysis of TILs for the incorporation of BrdU on day 13, combined with phenotyping for the expression of PD-1, LAG-3, and 4-1BB. Data pooled from 2 independent experiments. One-way ANOVA, n = 10. C and D, TILs were stained with SIY pentamer and analyzed for the expression of PD-1, LAG-3, and 4-1BB on day 13 after tumor injection. Representative data from more than 3 independent experiments, n = 5, one-way ANOVA. E, Tumors of mice bearing B16.SIY tumors for 13 days were analyzed for SIY pentamer-reactive cells and Ki-67 expression via flow cytometry, day 7, n = 8; day 13, n = 10; day 21, n = 5. Data pooled from 3 independent experiments. Two-way ANOVA.
- Figure 2.
CD8+ T cells undergo apoptosis in the tumor microenvironment. A, CD8+ cells from tumors and spleens of mice bearing B16.SIY tumors for 13 days were stained with SIY pentamer and analyzed for the presence of the active form of caspase-3 via flow cytometry. Data pooled from 3 independent experiments. Two-way ANOVA, n = 15. B, An autochthonous melanoma model driven by the melanocyte-specific, inducible expression of activated BRAF and deletion of PTEN was analyzed for the presence of active caspase-3 in CD8+ T cells from spleens and melanomas via flow cytometry. Data pooled from 2 independent experiments. Mann–Whitney U test. C, TILs from B16.SIY tumor-bearing mice were analyzed for active caspase-3 as in A and for expression of PD-1, LAG-3, and 4-1BB via flow cytometry. Data pooled from 6 independent experiments. One-way ANOVA, n = 29. D, TILs were stained with SIY pentamer and analyzed as in C, n = 29. Data pooled from 6 independent experiments. E, TILs from B16.SIY were isolated and analyzed for the expression of PD-1, LAG-3, and 4-1BB, as well as binding to fluorescently labeled Annexin V and Fixable Viability Dye eFluor 780, n = 10. Data pooled from 2 independent experiments. Two-way ANOVA. The indicated statistical differences are compared with the PD-1+LAG-3+4-1BB+ populations. F, TILs from mice bearing 13 day–established B16.SIY tumors were stained with SIY pentamer and analyzed for Ki-67 and active caspase-3 expression via flow cytometry, n = 10. Data pooled from 2 independent experiments, two-way ANOVA.
- Figure 3.
Decreased TIL apoptosis leads to increased tumor control. A, C57BL/6 mice were injected with 2 × 106 B16.SIY, 2 × 106 MC57.SIY, or 2 × 106 1969.SIY cells on day 0. On day 7, mice were sacrificed and analyzed for SIY-reactive TILs via flow cytometry. One-way ANOVA, B16.SIY, n = 5; MC57.SIY and 1969.SIY, n = 10. B, Similar conditions to Ki-67 expression of SIY-reactive TILs are shown (A). One-way ANOVA; B16.SIY, n = 5; MC57.SIY and 1969.SIY, n = 10. C, As in A, the fraction of SIY-reactive cells with active caspase-3 is shown. One-way ANOVA; B16.SIY, n = 5; MC57.SIY and 1969.SIY, n = 10. All data in A–C is pooled from 2 independent experiments. D, Bcl-xL or WT mice were injected with 2 × 106 B16.SIY cells on day 0. On day 13, mice were sacrificed and analyzed for active caspase-3 expression and to enumerate the number of TILs. Data pooled from 2 independent experiments. Mann–Whitney U test, n = 9. E, 2C or 2C Bcl-xL splenocytes were activated in vitro with plate-bound antibodies to CD3 and CD28 to generate activated effector CD8+ T cells. T cells (1 × 106) were transferred to mice bearing B16.SIY tumors on day 7 after tumor injection. No adoptive transfer, n = 10; 2C, n = 10; 2C Bcl-xL, n = 20. Data pooled from 2 independent experiments, two-way ANOVA.
- Figure 4.
4-1BB combination immunotherapy leads to tumor regression and TIL accumulation through an intratumoral process. A, C57BL/6 mice were subcutaneously inoculated with B16.SIY cells (2 × 106). Tumors were established for 7 days; then, cohorts were treated with either single antibodies against 4-1BB, CTLA-4, or PD-L1, or combinations of anti–4-1BB + anti–CTLA-4, or anti–4-1BB + anti–PD-L1. Antibodies were given intraperitoneally on days 7, 10, 13, and 16 after tumor injection. Each mouse received 100 μg of each indicated antibody at each time point, n = 10 mice per cohort. Data pooled from 2 independent experiments, two-way ANOVA. B, Combination immunotherapy was given as in A, but mice were sacrificed on day 13 and tumor-draining lymph nodes, spleens, and tumors were analyzed for SIY-reactive CD8+ T cells via flow cytometry. Data pooled from 2 independent experiments. One-way ANOVA, n = 10. C, C57BL/6 mice were injected subcutaneously with B16.SIY cells (2 × 106). Tumors were established for 7 days, at which point daily oral administration of FTY720 was begun. Mice received anti–4-1BB (100 μg) + anti–CTLA-4 (100 μg) or anti–4-1BB (100 μg) + anti–PD-L1 (100 μg) intraperitoneally on days 7, 10, 13, and 16 after tumor injection, n = 10. Data pooled from 2 independent experiments, two-way ANOVA. D, Blood from mice from C was analyzed at the endpoint of the experiment for circulating CD3+ cells using flow cytometry. E, C57BL/6 mice were injected subcutaneously with B16.SIY cells (2 × 106). Tumors were established for 7 days, at which point daily oral administration of FTY720 was begun. Mice received anti–4-1BB (100 μg) + anti–CTLA-4 (100 μg) or anti–4-1BB (100 μg) + anti–PD-L1 (100 μg) intraperitoneally on days 7, and 10 after tumor injection and were analyzed on day 13. The accumulation of SIY-reactive TILs was analyzed with flow cytometry. Data pooled from 3 independent experiments, n = 15, one-way ANOVA.
- Figure 5.
Anti–4-1BB combination immunotherapy decreases TIL apoptosis. A, C57BL/6 mice were inoculated subcutaneously with 2 × 106 B16.SIY cells and treated with immunotherapy on day 7 and day 10 after tumor cell injection. BrdU (0.8 mg/mouse) was administered intraperitoneally 24 hours before sacrifice of each cohort. On day 13 after tumor cell injection, the tumors were analyzed using flow cytometry to detect CD8+ T cells with incorporated BrdU; n = 9. Data pooled from 2 independent experiments. B, As in A, but Ki-67 expression was analyzed via flow cytometry. No treatment, n = 5; anti–4-1BB + anti–CTLA-4, n = 5; anti–4-1BB + anti–PD-L1, n = 4. C, As in A, but TILs were analyzed for the presence of active capase-3 via flow cytometry, data pooled from 4 independent experiments. No treatment, n = 19; anti–4-1BB + anti–CTLA-4, n = 20; anti–4-1BB + anti–PD-L1, n = 18; one-way ANOVA. D, As in A, but TILs were analyzed for the presence of active capase-3 and SIY reactivity via flow cytometry. Data pooled from 3 independent experiments. No treatment, n = 13; anti–4-1BB + anti–CTLA-4, n = 15; anti–4-1BB + anti–PD-L1, n = 14; one-way ANOVA.
- Figure 6.
TILs have increased DNA damage. A, Depicted are the top 10 enriched GO terms identified by DAVID from genes upregulated in PD-1+LAG-3+4-1BB+ TILs compared with PD-1+LAG-3−4-1BB− TILs, the −log(P value) of each, and the number of genes from the upregulated genes that were included the each GO term. B, Day 13 CD8+ cells from the spleens and tumors of mice were analyzed for γH2AX; n = 5, two-way ANOVA. C, TILs from day 13 tumors were analyzed for expression of PD-1, LAG-3, 4-1BB, and γH2AX; n = 10, one-way ANOVA. Data pooled from 2 independent experiments. D, Day 13 CD8+ cells were analyzed for expression of Ki-67 and γH2AX; n = 5, two-way ANOVA. E, C57BL/6 mice were injected subcutaneously with B16.SIY cells (2 × 106) on day 0. On days 7 and 10, mice were treated with 100 μg anti–4-1BB plus 100 μg anti–PD-L1. On day 13, CD8+ cells were analyzed for expression of Ki-67; n = 5, two-way ANOVA. F, The same CD8+ cells from E were also analyzed for γH2AX; n = 5, two-way ANOVA.
Additional Files
Supplementary Data
- Supplemental Figures and Legends 1-4 - Four supplemental figures and their legends.
Volume 6, Issue 1
